: Over the past two decades, microbeam technology has been a strong driver of new biological research. The proposed TR&Ds focus on three areas that collaborative users of the RARAF microbeams have identified as enabling significant advances in their research ? which in turn allow further technology developments: TR&D1: Cellular structures can currently be imaged and microbeam-targeted with 500 nm resolution: TR&D1 will allow imaging and microbeam-targeting of sub-cellular targets with 75 nm resolution. This development will open up a new class of experiments, involving targeting structures imaged with fluorescent proteins. TR&D2: Development of new microbeam-integrated single-cell analysis tools: single-cell gene-expression /single-cell capillary electrophoresis / single-cell Raman spectroscopy. TR&D3: Development of the most requested biology-support tools integrated within our microbeam systems: short- and long-term cell-environment-control tools, and a microfluidics-based microFACS fluorescence-activated cell sorter. In collaboration with investigators from both outside and within Columbia University, 24 Collaborative Projects (CPs) and 16 Service-based Projects (SPs) are proposed. Our TR&D projects are pushed by the needs of our CPs and in turn the results from the CPs drive further TR&D developments. SPs leverage from the extensive mature microbeam technologies already developed at RARAF. Training and Dissemination are central to the RARAF program. Ongoing training of users will be continued at all levels, from undergraduate to senior faculty. A new and highly successful annual 3-day Microbeam Training Course will be expanded; it involves formal lectures, and extensive hands-on experience. The corresponding on-line virtual microbeam training course, which consists of movies and podcasts synched with accompanying PowerPoint presentations, will correspondingly be expanded. Contemporary dissemination tools, such as wikis and LinkedIn, are now also used. In 2012 RARAF hosted the very successful and well attended 10th International Workshop on Microbeam Probes of Cellular Radiation Response. Dissemination of microbeam-related material through peer-reviewed publications and talks will be continued, both nationally and internationally.